| Background:RUNX1 is a key regulatory gene of human hematopoiesis,and have multiple splicing isoforms,including RUNX1b.RUNX1b has strong inhibitory effects on hematopoietic differentiation at the early stage of co-culture,and inhibits the transition from mesoderm to hemogenic endothelium.The inducible RUNX1b/hESC line had been established in our laboratory.Induction of RUNX1b from D0 severely prevents co-cultured cells from production of CD34+ population and its derived population.Compared with the control ones without induction the upregulated genes at in the D4 co-cultures that were induced RUNX1b overexpression from D0 were investigated their effects on hematopoiesis by us.Methods:RUNX1b/hESCs were co-cultured with AGM-S3,and induced or not induced RUNX1b overexpression from D0,and then the co-cultured cells are extracted RNA and performed deep sequencing analysis at D4.About 1500 genes were upregulated after RUNX1b overexpression,and five sgRNAs are designed per each gene so as to pool total 7500 sgRNAs to construct corresponding sgRNA knockout lentivirus library.This lentivirus library transduced RUNX1b/hESCs to construct the knockout hESC mutation library,and then amplify the library and freeze at-80℃.The knockout hESC mutation library is co-cultured with AGM-S3 to induce hematopoietic differentiation,and then three hematopoietic populations,including hematopoietic stem/progenitor cells,erythroid directed cells and myeloid directed cells,are sorted at D14.The genome DNAs were extracted from the knockout hESC mutation library and three hematopoietic differentiation populations.The sgRNA fragments inserted in genome were amplified by PCR and performed deep sequencing.Compared with the knockout hESC mutation library the genes having significantly increased insertion frequency of sgRNA in hematopoietic differentiation populations will be regarded as candidate genes that might be closely related to the inhibitory effects of RUNX1b overexpression on hematopoietic differentiation in the early stage.Results:Compared with the knockout hESC mutation library,the sgRNA insertion frequencies of IRX2,GABRQ and CACNG5 increase significantly in hematopoietic stem/progenitor cells,myeloid directed cells and erythroid directed cells.The sgRNA insertion frequency of IGSF5 increase significantly in hematopoietic stem/progenitor cells and myeloid directed cells.The sgRNA insertion frequency of SLC32A1 increase significantly in hematopoietic stem/progenitor cells and erythroid directed cells.According further verification it is found that when these 5 genes are consistently knockdown during the stages of both hESC culture and co-culture the efficiency of hematopoietic differentiation was promoted significantly.Among them,the consistent overexpression of CACNG5 or IGSF5 during the stages of both hESC culture and co-culture significantly decreased the efficiency of hematopoietic differentiation respectively,which effects may be closely related to the change of cell cycle status.IGSF5 knockout significantly promoted hematopoietic differentiation efficiency,which is similar to the effects caused by its knockdown. |
